Chlamydia trachomatis is the leading cause of preventable blindness (~8 million visually impaired people worldwide) and bacteria-associated sexually transmitted diseases (STD) with over 900,000 reported cases of STD occurring each year in the United States alone. While chlamydial STD infections in males are typically symptomatic, women often have asymptomatic infections resulting in infertility or life threatening ectopic pregnancies. Clinical treatment failure and demonstration of in vitro resistance to select antibiotics suggests that drug resistance to current therapeutics is possible, making research that could both identify new drug targets and lead to vaccines crucial to public health. Essential metabolic pathways in bacteria are common drug targets and metabolic pathway mutants also have shown promise as live-attenuated vaccine strains. The shikimate pathway (referred to in this application as the chorismate pathway [CP]) produces chorismate, which is required for the de novo synthesis of essential aromatic compounds. Because the CP is present in bacteria, but absent in mammals, enzymes of the CP have been used as targets for antibiotic development and CP mutants have been constructed for use as live-attenuated vaccines. While the genes comprising the CP pathway are predicted to be present in the Chlamydia genomes, they have yet to be functionally validated and chorismate-utilizing gene homologs are absent in C. trachomatis. Subsequently, this application will test the hypothesis that C. trachomatis uses the CP to produce chorismate for aromatic compound synthesis by: 1) functionally analyzing key pathway enzymes (AroG, AroDE, and AroA) using genetic and biochemical approaches, including complementation studies in Escherichia coli null mutants, enzyme assays using purified recombinant proteins, and inhibitor assays;2) detecting chorismate from C. trachomatis bacterial extracts using mass spectroscopy;and, 3) determining how chorismate is utilized by C. trachomatis using radio-label tracing experiments and genome library screens. Successful completion of this application will fill in gaps in our understanding of chlamydial metabolism, identify new drug targets to inhibit chlamydial growth, and will identify gene knockout candidates to create attenuated strains. Chlamydia is the number one cause of bacterial infections in the United States and the leading cause of blindness and bacterial STD worldwide. This research application seeks to identify new ways to treat and prevent chlamydial infections, as well as to elucidate unknown features of the chlamydial metabolism.